CN107365979B - Attachment of nano objects to beam deposition structures - Google Patents

Abstract

The beam-induced deposition decomposes the precursor at a precise location on the surface. The surface is treated to provide a linking group on the surface of the deposit, and the sample is treated to attach nano-objects to the linking group. The nano-objects are useful in a variety of applications. When a charged particle beam is used to decompose a precursor, the charged particle beam can be used to form an image of the surface to which the nano-objects are attached.

Description

Attachment of nano objects to beam deposition structures

Cross Reference to Related Applications

This application claims priority from U.S. provisional application 62/335,423 filed on 12/5/2016 and U.S. application 15/188,862 filed on 21/6/2016, both of which are hereby incorporated by reference.

Technical Field

The present invention relates to attachment of nano objects to surfaces, and in particular to formation of attachment sites using beam induced deposition.

Background

Many applications, including biomaterial sensing, photonics, surface plasmon photonics, and quantum information processing, require the assembly of nano-objects into macroscopic arrays. Several top-down assembly methods have been developed, including photolithography or dip-pen techniques, as well as bottom-up methods (using patterned self-assembled monolayers or electrostatic self-assembly). While these methods are capable of patterning nano-object arrays at high resolution, the assembled parts are only weakly bonded to the substrate and therefore cannot undergo further processing, such as wet chemical processing steps (sonication) or subsequent photolithography. Such further processing is often required for device applications where nano-objects act as active ingredients in microfluidic devices, as sensing probes or photon sources where they are coupled to plasmonic structures or other optical elements.

The selective adsorption of the nano-objects by biomolecules to the functionalized surface can be used for sensing of biomolecules. Gold is generally the substrate of choice for biosensing due to its inertness relative to biological systems and its ability to form mercapto or thiol terminated surfaces (-SH). These thiol groups can spontaneously form disulfide bonds with other thiol-terminated molecules. Historically, these thiol-terminated biofunctional surfaces have been formed by functionalizing the gold surface with thiol groups, followed by the introduction of alkylthioalkyl (R-SH) groups, which spontaneously order and form self-assembled monolayers with terminals opposite to the thiol groups functionalized with bioselective groups, such as enzymes or biological substrates. Then, an analyte can be added that selectively binds to the functionalized monolayer.

Self-assembled monolayers have been proposed due to the possibility of impacting the structure of biomolecules of interest on the surface of the bulk matrix. For example, if such a monolayer is not used, proteins may be irreversibly denatured by interaction with a hydrophobic or hydrophilic functionalized surface. Thus, if secondary, tertiary or quaternary structure is important, direct immobilization on the surface is not desirable.

The gold required for the introduction of thiol groups is usually applied using photolithographic techniques, which are time consuming and expensive. A method for immobilizing biomolecules on a surface without the need for intermediate steps of gold application and self-assembled monolayer formation is desirable.

In general, a robust and accurate method of attaching nano-objects to a surface at precise locations is desired.

Disclosure of Invention

It is an object of the present invention to provide a system for attaching nano-objects to a surface at precise locations.

Beam-induced deposition decomposes the precursor at a precise location on the surface. The sample is treated to provide a linking group on the surface of the deposit, and the sample is treated to attach the nano-objects to the linking group.

The foregoing has outlined rather broadly the features and technical advantages of the present invention in order that the detailed description of the invention that follows may be better understood. Additional features and advantages of the invention will be described hereinafter. It should be appreciated by those skilled in the art that the conception and specific embodiment disclosed may be readily utilized as a basis for modifying or designing other structures for carrying out the same purposes of the present invention. It should also be realized by those skilled in the art that such equivalent constructions do not depart from the scope of the invention as set forth in the appended claims.

Drawings

For a more complete understanding of the present invention and the advantages thereof, reference is now made to the following descriptions taken in conjunction with the accompanying drawings, in which:

FIG. 1 is a flow chart illustrating a method for nano-object attachment;

FIG. 2A is a photomicrograph showing a top view of a deposit; and fig. 2B is a photomicrograph showing a side view of the deposit;

FIG. 3 is a photomicrograph showing a pattern of deposits, and the inset shows a single deposit with attached nano-objects;

FIG. 4 is a flow chart illustrating a method of nano-object attachment;

FIG. 5 is a flow chart illustrating a method of attaching multiple types of nano-objects;

FIG. 6 shows a surface with multiple types of nano-objects attached;

FIG. 7 shows a flow diagram of another method of nano-object attachment;

FIG. 8 illustrates a prior art method of attachment of nano objects;

FIG. 9 shows a nano object attached to a substrate; and

fig. 10 schematically illustrates a dual beam system suitable for attaching nano-objects to a substrate.

Detailed Description

Embodiments of the present invention provide a method and apparatus for precise selective attachment of nano objects to specific regions on a substrate.

Attachment of the nano-objects to the substrate surface is achieved using beam induced deposition by: depositing a material on the substrate, modifying the surface properties of the deposit, and then attaching the nano-objects to the modified deposit surface. The method provides the option of nano-objects only attaching to the modified surface of the deposit and not to the substrate where the deposit was not prepared. The nano objects typically have critical dimensions in the range of 1nm to 1000 nm. The critical dimension may be, for example, the diameter of a nanodot, a crystal, a wire, a tube, a sheet, or a flake. The attached nano-objects may be individual nano-objects or clusters consisting of multiple nano-objects, such as multiple nanodots, nanocrystals, nanowires, or sheets.

Fig. 1 is a flow chart illustrating a method of attaching nano objects to a substrate. In optional step 101, the surface is preferably examined with charged particle beam imaging. At step 102, a deposit is formed on the surface of the substrate by beam-induced deposition. Deposition of the various materials may be carried out using well known suitable deposition precursors. Fig. 2A and 2B show top and side views, respectively, of a deposit formed using electron beam induced deposition. At step 104, the surface chemistry of the deposit is typically modified with a functionalization precursor. Preferably, the functionalization precursor only affects beam-induced deposits, such that the entire substrate can be exposed to the functionalization precursor and only the beam-induced deposits will be modified.

At step 106, one or more nano-objects are attached to the modified deposition surface. Because the nano-objects only adhere to the chemically modified surface of the deposited material, the entire substrate may be exposed to the nano-objects, for example by immersing the substrate in a bath or exposing the substrate to a gas, and the nano-objects will only attach to the beam-induced deposits. Thus, the accuracy of positioning of the nano-objects on the substrate is determined by the accuracy of beam-induced deposition, and the functionalization and nano-object attachment process can be applied to the entire workpiece. Attaching the nano-objects may optionally be a multi-step process, wherein a first nano-object subsequently captures a second nano-object. In some embodiments, the functionalized deposition is capable of forming covalent bonds with the nano objects. Attachment of the nano-objects by covalent bonding provides a robust attachment that is stronger than by electrostatic interaction or physical fixation. The covalent bonds may include, for example, disulfide bonds, sulfonamide bonds, and/or phosphonamide bonds, although the invention is not limited to these bonds.

At optional step 108, an image is formed from the surface having the nano-objects. For example, if beam-induced deposition is performed with a charged particle beam, a charged particle beam image (such as produced by scanning electron beam (SEM) imaging or ion beam imaging) may be used. Fig. 3 shows an SEM image of a substrate surface 302 having a pattern of deposits 304. Inset 308 shows an enlarged view of a single deposit 304 with attached nano-objects 306. In the examples of fig. 2A, 2B and 3, the deposit is carbon and the attached nano objects are nanodiamonds. It should be noted that the nanodiamonds did not attach to the substrate surface where the deposits had not yet formed, thereby showing selective attachment of the nanodiamonds to the modified deposits. At step 110, the work piece with attached nano objects is applied to a useful result, such as sensing biomolecules or acting as an information processing element.

Fig. 4 is a flow chart of a method of attaching nano objects to carbon deposited on a substrate by beam-induced deposition. In optional step 401, the surface is inspected in a vacuum chamber by charged particle beam imaging. At step 402, a carbon-bearing precursor gas is introduced into the vacuum chamber and adsorbed onto the substrate surface. Many different carbon-bearing precursor gases, such as naphthalene, ethylene, or styrene vapors, can be used to deposit carbon. In step 404, the surface of the substrate is irradiated with a charged particle beam to decompose the precursor gas to form a solid carbon deposit on the surface of the substrate. Typically, this deposition process results in amorphous carbon deposition, however, other forms of carbon deposition are possible. Due to the precision of charged particle beam processing, complex nanoscale deposits can be accurately formed using this method. The process is preferably carried out in a vacuum chamber of a charged particle beam apparatus. The charged particle beam is typically an electron beam, but an ion beam or a cluster beam may also be used. Laser beams may also be used to decompose the precursor.

In step 406, the substrate surface with the deposited material is exposed to a solution containing ammonia (NH)₃) The plasma of (2). NH (NH)₃The plasma modifies the surface chemistry of the carbonaceous deposits resulting in amine groups (-NH)₂) And/or amide groups (-CONH)₂) Formation on the surface of carbon deposits. The substrate may be processed in the same charged particle apparatus in which the deposits are formed, or the substrate may be removed and may undergo plasma processing in a different apparatus.

After surface modification, nano objects are attached to the modified deposit surface at step 408. For example, nanodiamonds having a capped carboxylic acid group (-COOH) can be bonded to the modified deposit surface by coupling with a coupling agent such as EDC (1-ethyl-3- (dimethylaminomethyl) carbodiimide). Such coupling covalently bonds the nanodiamond to the modified deposit through formation of amide bonds.

Because of-NH required for amide bond formation with-COOH containing nanodiamonds₂Groups are only present on the surface of the modified deposit, so nanodiamond attachment only occurs on such surfaces. This property allows the entire sample substrate to be exposed to the nano objects, rather than limiting the application of the nano objects. For example, the sample substrate may be immersed in a solution containing EDC and nanodiamonds, with attachment occurring only at sites where amide-terminated deposits are located thereon.

Furthermore, deposition within the vacuum chamber of the charged particle device allows the charged particle beam imaging to be synchronized with the deposition process. Charged particle beam imaging is performed by scanning a charged particle beam while detecting secondary electrons or backscattered electrons, the brightness of each point on the image being determined by the number of detected secondary particles. This allows for careful control of the location of the deposits and also allows metrology to be performed during deposition. At optional step 410, the electron beam is scanned across the surface and an SEM image is formed from the surface with the nano-objects attached. The SEM image may be formed during any part of the process.

Attachment of the nano-objects by covalent bonding provides a robust attachment that is stronger than by electrostatic interaction or physical fixation. Indeed, after 12 hours of sonication, 90% of the attached nanodiamonds remained attached to the substrate surface, indicating a strong attachment.

Although the method of fig. 4 functionalizes the surface with amide groups, other chemical functionalizations are also possible by using different functionalization precursors. For example, various precursors may be used to form a surface functionalized with-OH, or may form-C ═ O, -COOH, -NO2, -SO2, -PO3, halide, -SH, or-C ═ S terminations. Alternative multi-step attachment methods may also be used. For example, functionalizing the surface may be performed after attaching a protein-specific antibody to the functionalized surface, which binds to the protein of interest, thereby immobilizing it at the desired site on the substrate.

At step 412, the nano-objects are used for practical applications. For example, a sensor may be formed by attaching a dye or other chromophore to the surface of a deposit, in which a target molecule is present that initiates a color change. Alternatively, a molecule specific sensor may be formed by forming a deposit on a pair of electrodes and forming a conductive surface via functionalization with polar groups. As the target molecule interacts with the surface, the conductivity between the conductors will change and can be read as a signal.

By forming a functionalized deposit of a first surface chemistry, and then forming a new functionalized deposit having a different surface chemistry, a single substrate can have multiple functionalities applied to different regions or array formations of, for example, a TEM grid.

Similarly, the single-function effect can be applied repeatedly, for example, to form a series of first deposits and bind first protein-specific antibodies to all available sites, and then to form second deposits and bind second protein-specific antibodies to all available sites. In this case, since the only available sites for each step are those not occupied by the previous step, the same deposition and functionalization chemistry can be reused, allowing the localization of the desired bonding sites for a variety of different target molecules. Alternatively, different depositions and/or functionalizations may be used sequentially.

Fig. 5 is a flow chart of a repetitive deposition and attachment process. At step 502, a deposit is formed on a surface of a substrate. At step 504, the surface chemistry of the deposit is modified. Next, one or more nano-objects are attached to the modified deposit surface at step 506. At step 508, it is determined whether additional nano-objects are attached, which are different from those to which step 506 is attached. If a different nano-object is to be attached, the method returns to step 502. If not, the method ends at block 510. The composition of the deposit or modification of the surface of the deposit may be the same or different than that applied in the previous step, depending on the nano-objects to be attached in the current cycle. Fig. 6 shows a substrate 602 having a plurality of different nano- objects 604, 606, and 608 attached to a surface by subsequent deposition cycles.

The deposition, functionalization and attachment steps are applicable to biomolecule analysis. In this embodiment, the attached receptive rice organism is a biomolecule, such as a protein or an antibody. Charged particle deposition provides the ability to form high aspect ratio structures, including those with sulfur-containing components. The direct deposition of such structures allows for attachment of fragile biomolecules to the ends of the structures, avoiding interaction between the biomolecules and the underlying substrate, while also avoiding the intermediate steps of gold lithography and subsequent monolayer formation used in the prior art.

Referring to fig. 7, a method of deposition of sulfur-containing deposits and subsequent functionalization and attachment of biomolecules is shown. At step 702, a supporting sulfur precursor gas is adsorbed on the surface of a sample substrate. The sample substrate is then irradiated with a charged particle beam at step 704, resulting in the decomposition of the supported sulfur precursor and the formation of a sulfur-containing deposit.

Some precursor chemistries offer the possibility of sulfur deposition, such as sulfur hexafluoride, methane sulfophenamine, methyl mercaptan, methyl disulfide, and methyl methane sulfonic acid. Other suitable supported sulfur precursors are known and available.

Although fig. 4 depicts the deposition of a carbon-containing material and fig. 7 depicts the deposition of a sulfur-containing material, other materials may be used. For example, phosphorus may be deposited by supporting a phosphorus precursor, such as platinum tetra-trifluorophosphate. Although the material deposited by decomposition of platinum tetra-trifluorophosphate comprises platinum, it also contains a significant amount of phosphorus. The choice of functionalization may be different or the same for deposits having different compositions. NH (NH)₃Application of the plasma results in amine and amide bonds on the surface of the deposited carbon. Sulfonamide linkage (-SO) can be formed on the surface of sulfur₂-NH₂) And a phosphonamide bond (-PO-NH) can be formed on the surface of phosphorus₂). Nanodiamonds, nanoparticles, biomolecules, quantum dots, and other nano-objects may be attached to the phosphorus-containing deposits.

At step 706, the sample is treated to functionalize the surface of the deposit, thereby forming a thiol group termination. Sample processing may also occur simultaneously with the formation of the sulfur-containing deposits, thereby combining steps 704 and 706. This may be achieved by co-deposition with ammonia or post-deposition treatment with ammonia, thermally cracked hydrogen or other suitable reducing agent.

After the treatment to convert surface sulfur to thiol groups, biomolecules can now be attached to the sulfur-containing structure, for example by the formation of disulfide bonds between-SH containing amino acid residues in the enzyme. In addition, it is common to engineer biomolecules such that one of these residues is located at a desired site along the biomolecule, such that the bonding occurs at an artificially designed site within the biomolecule.

The use of direct deposition of sulfur-containing nanostructures circumvents the time-consuming step of gold photolithography, allowing direct attachment of biomolecules to the substrate. Furthermore, the direct deposition process may be performed on a variety of materials for which conventional gold photolithography methods are not possible or practical.

Fig. 8 shows a prior art method of attaching biomolecules 808 to a substrate 802. The gold layer 804 is first applied by photolithography, followed by the formation of the self-assembled monolayer 806. The biomolecules 808 are then attached to the ends of the monolayer molecules. The biomolecules are separated from the substrate by the monolayer and therefore do not interact with the substrate.

Fig. 9 shows a biomolecule 906 bonded to the end of a sulfur nanorod 904. The nanopillars extend from the surface of the substrate 902. Similar to the prior art method of fig. 8, the biomolecules are separated from the substrate and do not undergo interaction with the substrate.

Indirect attachment of biomolecules to sulfur-containing structures may also be performed. For example, the enzyme may be immobilized on the end of the structure, allowing for the binding of other molecules while keeping them far enough away from the surface not to disrupt the secondary, tertiary or quaternary structure.

Although much of the above discussion relates to thiol or sulfhydryl termination of sulfur-containing structures, one skilled in the art will recognize that other materials and terminating functional groups may be applied for attachment of biomolecules to a substrate surface. For example, a mercapto-terminated or carbon structure may be used.

The attachment of the desired biomolecules to the direct deposition structure allows additional flexibility in the choice of substrates on which the biomolecules can be immobilized. In addition, the direct deposition process allows for repeated deposition and attachment to be performed, resulting in a base with some different biomolecules attached to a single substrate. Such repeated deposition and attachment avoids repeated photolithography steps that would potentially damage previously attached biomolecules.

In some embodiments, the deposition precursor is supplied as a gaseous precursor directed at the substrate surface by a gas injection system within a vacuum chamber of the charged particle beam apparatus. In other embodiments, the deposition precursor supply is a liquid. In other embodiments, the precursor gas is disposed within the housing of a vacuum chamber (sometimes referred to as an environmental cell). If multiple deposition and attachment cycles are performed, the substrate may be removed from the vacuum chamber of the charged particle beam apparatus between subsequent cycles, or any number of a series of cycles may be performed without removal.

Fig. 10 illustrates an exemplary dual beam system 1002 for implementing an embodiment of the present invention. Suitable dual beam systems are commercially available from FEI Company of hilsburr, Oregon, for example (the assignee of the present invention). Although examples of suitable hardware are provided below, the invention is not limited to implementation in any specific type of hardware.

The dual beam system 1002 has a vertically mounted electron beam column 1004 and a focused particle beam (FIB) column 1006, the focused ion beam column 1006 being mounted on an evacuable specimen chamber 1008 at an angle of about 52 degrees from vertical. The specimen chamber may be evacuated by a pump system 1009, the pump system 1009 typically comprising one or more or a combination of turbomolecular pumps, oil diffusion pumps, ion getter pumps, vortex pumps, or other known pumping devices.

The electron beam column 1004 includes an electron source 1010 (such as a schottky emitter or a cold field emitter) for generating electrons and electron optical lenses 1012 and 1014 that form a finely focused electron beam 1016. The electron source 1010 is typically held at a potential between 500V and 30kV above the potential of the workpiece 1018, which is typically held at ground potential.

Thus, the electrons impact the workpiece 1018 with a landing energy of approximately 500eV to 30 keV. A negative potential may be applied to the workpiece to reduce the landing energy of the electrons, which reduces the interaction volume of the electrons with the workpiece surface, thereby reducing the size of the nucleation sites. The workpiece 1018 may comprise, for example, a semiconductor or other material to which the nano-objects are to be attached. The point of impact of the electron beam 1016 may be located on the surface of the workpiece 1018 and scanned over the surface of the workpiece 1018 by the deflection coils 1020. The operation of the lenses 1012 and 1014 and the deflection yoke 1020 is controlled by a scanning electron microscope power source and control unit 1022. The lenses and the deflection unit may use an electric field, a magnetic field, or a combination thereof.

The workpiece 1018 is on a movable stage 1024 of the specimen chamber 1008. The gantry 1024 is preferably movable in a horizontal plane (X and Y axes) and vertically (Z axis), and can tilt about sixty (60) degrees and rotate about the Z axis. The door 1027 may be opened for inserting the workpiece 1018 onto the X-Y-Z gantry 1024 and also for servicing an internal gas supply reservoir (not shown), if used. The door is interlocked so that it cannot be opened if the specimen chamber 1008 is evacuated. Gantry 1024 can be cooled, such as by a Peltier cooler (not shown), or heated, such as by a resistive heater 1026.

Mounted on the vacuum chamber are a plurality of Gas Injection Systems (GIS)1030 (two shown). Each GIS includes a reservoir (not shown) for holding precursor or activation material and a needle 1032 for directing gas to the surface of the workpiece. Each GIS also includes means 1034 for regulating the supply of precursor material to the workpiece. In this example, the adjustment means is described as an adjustable valve, but the adjustment means may also comprise, for example, an adjustment heater for heating the precursor material to control the vapor pressure. An optional plasma generator 1039 generates a plasma for exposing the workpiece 1018 to the plasma for processing.

When electrons in the electron beam 1016 strike the workpiece 1018, secondary electrons, backscattered electrons, and auger electrons are emitted and may be detected to form an image or to determine information about the workpiece. The secondary electrons are detected, for example, by a secondary electron detector 1036 (such as an Everhard-Thornley detector) or a semiconductor detector device capable of detecting low energy electrons. The signals from the detector 1036 are provided to a system controller 1038, and the system controller 1038 controls a monitor 1040, the monitor 1040 being used to display user controls and an image of the workpiece using the signals from the detector 1036.

The chamber 1008 is evacuated by the pump system 1009 under the control of the vacuum controller 1041. The vacuum system provides about 3x 10 within the chamber 1008^-6Vacuum of mbar. When a suitable precursor or activator gas is introduced onto the sample surface, the chamber background pressure can typically be raised to about 5x 10^-5mbar. The local pressure and gas concentration at the surface of the workpiece are significantly greater than the background pressure and concentration.

The focused ion beam column 1006 includes an upper neck portion 1044, with an ion source 1046 and a focusing column 1048 (including an extractor electrode 1050) and an electrostatic optical system (including an objective lens 1051) located within the upper neck portion 1044. The ion source 1046 may comprise a liquid metal gallium ion source, a plasma ion source, a liquid metal alloy source, or any other type of ion source. The axis of the focusing column 1048 is tilted 52 degrees from the axis of the electron column. An ion beam 1052 passes from an ion source 1046 between a focusing column 1048 and an electrostatic deflector 1054 toward a workpiece 1018.

The FIB power source and control unit 1056 provides the potential at the ion source 1046. The ion source 1046 is typically maintained at a potential between 1kV and 60kV above the potential of the workpiece, and the workpiece 1018 is typically maintained at ground potential. Thus, the ions impact the workpiece at landing energies of about 1keV to 60 keV. The FIB power source and control unit 1056 is coupled to a deflection plate 1054, which deflection plate 1054 can cause the ion beam to outline a corresponding pattern on the upper surface of the workpiece 1018. In some systems, the deflection plate is placed before the final lens, as is well known in the art. Beam blanking electrodes (not shown) within ion beam focusing column 1048 cause ion beam 1052 to impact on a blanking aperture (not shown) instead of workpiece 1018 when a FIB power source and control unit 1056 applies a blanking voltage to the blanking electrodes.

The ion source 1046 generally provides a beam of singly-charged gallium positive ions that may be focused into a one-tenth of a sub-micron wide beam at the workpiece 1018 for modifying the workpiece 1018 by ion milling, enhanced etching, material deposition, or for imaging the workpiece 1018.

A system controller 1038 controls the operation of the various components of the dual beam system 1002. Through the system controller 1038, a user, through commands entered into a conventional user interface (not shown), can cause the ion beam 1052 or the electron beam 1016 to scan in a desired manner. Alternatively, the system controller 1038 may control the dual beam system 1002 according to programmed instructions. Fig. 10 is a schematic diagram, does not include all elements of a typical dual beam system, and does not reflect the actual appearance and size of all elements or the relationship therebetween.

There are many novel aspects to the preferred methods or apparatus of the present invention and, because the invention may be embodied in different methods or apparatus for different purposes, each embodiment need not present every aspect. Moreover, many aspects of the described embodiments may be separately patentable. The present invention has broad applicability and can provide many of the benefits described and illustrated by the examples above. The embodiments will vary greatly depending upon the particular application, and not every embodiment will provide all of the benefits and meet all of the objectives achievable by the present invention.

In one embodiment, a method of attaching nano objects to a sample substrate is presented, the method comprising:

providing a deposition precursor to a surface of a substrate located within a vacuum chamber of a charged particle apparatus;

directing a beam at the substrate, the beam decomposing the deposition precursor to form a deposit on the surface of the substrate at a location impacted by the beam;

treating the sample to provide attachment groups on the surface of the deposit; and

the sample is treated to chemically bond the nano-objects to the linking groups.

In some embodiments, directing the beam at the substrate comprises directing a charged particle beam at the surface.

In some embodiments, a charged particle beam image of the surface of the attached nano-object is formed.

In some embodiments, the chemical bond comprises a covalent bond. In some other embodiments, the covalent bond comprises an amide bond, a disulfide bond, a sulfonamide bond, or a phosphonamide bond.

In some embodiments, the nano objects comprise nanodiamonds or biomolecules.

In some embodiments, the nano objects have a critical dimension in a range between 1nm and 1000 nm. In some other embodiments, the nano-objects comprise nano-dots, crystals, tubes, sheets or flakes.

In some embodiments, the nano-objects comprise clusters of individual nano-objects. In some other embodiments, the nano-objects comprise clusters of nano-dots, crystals, wires, tubes, sheets, or flakes.

In some embodiments, depositing the precursor comprises carrying a carbon precursor, and the deposit comprises carbon.

In some embodiments, depositing the precursor includes carrying a sulfur precursor, and the deposit includes sulfur.

In some embodiments, depositing the precursor comprises carrying a phosphorous precursor, and depositing comprises phosphorous.

In some embodiments, the deposits have an aspect ratio greater than 1.

In some embodiments, the nano-objects are bonded at the end of the deposit distal to the sample substrate.

In some embodiments, the deposition is sufficiently high to prevent bonding of the nano-objects from interacting with the sample substrate.

In some embodiments, processing the sample to provide the linking group occurs within a vacuum chamber of the charged particle apparatus. In other embodiments, the processing occurs outside of the vacuum chamber.

In some embodiments, treating the sample to provide the linking group comprises plasma treatment. In some other embodiments, the plasma treatment comprises treatment with an ammonia plasma. In some other embodiments, the plasma treatment includes treating the sample with a liquid. In some embodiments, the liquid is an acid.

In some embodiments, providing the linking group on the surface of the deposit occurs simultaneously with the formation of the deposit.

In some embodiments, the processing is performed with a charged particle beam and a precursor gas mixture consisting of a deposition precursor gas and a linker precursor gas. In some embodiments, the linker precursor gas is ammonia. In some embodiments, the charged particle beam is an electron beam, a laser beam, or an ion beam.

In some embodiments, processing the sample to chemically bond the nano-objects includes processing the sample with a liquid containing the nano-objects.

In some embodiments, the nano-objects undergo a surface treatment prior to bonding to the linking group. In some embodiments, the surface modification comprises functionalizing the surface of the nano-objects with carboxylic acid groups.

Although most of the previous description relates to the attachment of nanodiamonds and proteins, the present invention may be used to attach nanoscale objects of any suitable material. The terms "workpiece," "sample," "substrate," and "specimen" are used interchangeably in this application unless otherwise indicated. Similarly, the terms "deposit" and "deposit" are used interchangeably and refer to solid material that remains on a surface after a deposition process. In addition, whenever the terms "automatic," "automated," or similar terms are used herein, these terms should be understood to include manual initiation of an automated or automated process or step.

In the following discussion and in the claims, the terms "include" and "comprise" are used in an open-ended fashion, and thus should be interpreted to mean "including, but not limited to. If any term is not explicitly defined in the specification, it means that the term is given its plain and ordinary meaning. The accompanying drawings are intended to aid in the understanding of the invention and, unless otherwise indicated, are not drawn to scale.

The various features described herein may be used in any combination or subcombination of functions and not only those combinations described in the embodiments herein. Accordingly, the disclosure should be construed to provide written description of any such combination or sub-combination.

Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made herein without departing from the scope of the invention as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, composition of matter, means, methods and steps described in the specification. As one of ordinary skill in the art will readily appreciate from the disclosure of the present invention, processes, machines, manufacture, compositions of matter, means, methods, or steps, presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein may be utilized according to the present invention. Accordingly, the appended claims are intended to include within their scope such processes, machines, manufacture, compositions of matter, means, methods, or steps.

Claims (26)

1. A method of attaching a nano object to a sample substrate, the method comprising:

providing a deposition precursor to a surface of the substrate, the substrate being located within a vacuum chamber of a charged particle beam apparatus;

directing a beam at the substrate, the beam decomposing a deposition precursor to form a deposit on a surface of the substrate at a location impacted by the beam;

treating the sample by exposing the surface of the sample to plasma to provide attachment groups on the surface of the deposit; and

treating the sample to chemically bond the nano-objects to the linking groups.

2. The method of claim 1, wherein directing a beam at the substrate comprises directing a charged particle beam at the surface.

3. The method of claim 2, further comprising forming a charged particle beam image having the surface with attached nano-objects.

4. The method of any one of claims 1 to 3, wherein the chemical bond comprises a covalent bond, an amide bond, a disulfide bond, a sulfonamide bond, or a phosphonamide bond.

5. A method according to any one of claims 1 to 3, wherein the nano objects comprise nanodiamonds or biomolecules.

6. The method of any one of claims 1 to 3, wherein the nano objects have a critical dimension in a range between 1nm and 1000 nm.

7. The method of any one of claims 1 to 3, wherein the nano-objects comprise nanodots, crystals, wires, tubes, sheets or flakes, or clusters of individual nano-objects.

8. The method of any of claims 1-3, wherein the deposition precursor comprises a supported carbon precursor and the deposit comprises carbon.

9. The method of any one of claims 1 to 3, wherein the deposition precursor comprises a supported sulfur precursor and the deposit is substantially sulfur.

10. The method of any of claims 1 to 3, wherein the depositing precursor comprises carrying a phosphorus precursor and the deposit is substantially phosphorus.

11. The method of any one of claims 1 to 3, wherein the deposit has an aspect ratio greater than 1.

12. The method of any one of claims 1 to 3, wherein the nano-objects are bonded at an end of the deposit distal to the sample substrate.

13. The method of claim 12, wherein the deposit is sufficiently high to prevent the bonded nano-objects from interacting with the sample substrate.

14. The method of any one of claims 1 to 3, wherein processing the sample to provide a linking group occurs within the vacuum chamber of the charged particle beam apparatus.

15. The method of any one of claims 1 to 3, wherein processing the sample to provide a linking group occurs outside the vacuum chamber of the charged particle beam apparatus.

16. A method according to any one of claims 1 to 3, wherein the plasma comprises an ammonia plasma.

17. The method of any one of claims 1 to 3, wherein treating the sample to provide a linking group comprises plasma treating the sample with a liquid.

18. The method of claim 17, wherein the liquid is an acid.

19. The method of any one of claims 1 to 3, wherein providing a linking group on the surface of the deposit occurs simultaneously with the formation of the deposit.

20. The method of any one of claims 1 to 3, wherein processing the sample to provide linker groups on the surface of the deposit is performed with a charged particle beam and a precursor gas mixture, the precursor gas mixture consisting of a deposition precursor gas and a linker precursor gas.

21. The method of any one of claims 1 to 3, wherein treating the sample to provide linker groups on the surface of the deposit is achieved by using ammonia vapor as a linker precursor gas.

22. The method of claim 2, wherein directing a beam of charged particles comprises directing an electron beam or an ion beam.

23. The method of any one of claims 1 to 3, wherein the beam is a laser beam.

24. The method of any one of claims 1 to 3, wherein processing the sample to chemically bond nano objects comprises processing the sample with a liquid containing nano objects.

25. The method of any one of claims 1 to 3, wherein the nano objects undergo surface modification prior to bonding to the linking group.

26. The method of claim 25, wherein the surface modification comprises functionalizing the surface of the nano-objects with carboxylic acid groups.

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